Redox reversible bipyridyl-osmium complex conjugates

ABSTRACT

Novel bipyridyl-osmium complex conjugates and their use in electrochemical assays are described. The redox reversible-osmium complexes can be prepared to exhibit unique reversible redox potentials and can thus be used in combination with other electroactive redox reversible species having redox potentials differing by at least 50 millivolts in electrochemical assays designed for use of multiple electroactive species in the same cell and in the same sample without interference between the two or more redox coupled conjugate systems.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/322,791, filed May 28, 1999, now U.S. Pat. No. 6,352,824, whichclaims priority under 35 U.S.C. § 119(e) to U.S. Provisional Ser. No.60/087,576, filed Jun. 1, 1998, which is expressly incorporated byreference.

FIELD OF THE INVENTION

This invention relates to novel redox-reversible conjugates. Moreparticularly the invention is directed bipyridyl complexed-osmiumconjugates useful for detection and quantification of biologicallysignificant analytes in a liquid sample.

BACKGROUND AND SUMMARY OF THE INVENTION

Therapeutic protocols used today by medical practitioners in treatmentof their patient population requires accurate and convenient methods ofmonitoring patient disease states. Much effort has been directed toresearch and development of methods for measuring the presence and/orconcentration of biologically significant substances indicative of aclinical condition or disease state, particularly in body fluids such asblood, urine or saliva. Such methods have been developed to detect theexistence or severity of a wide variety of disease states such asdiabetes, metabolic disorders, hormonal disorders, and for monitoringthe presence and/or concentration of ethical or illegal drugs. Morerecently there have been significant advancements in the use ofaffinity-based electrochemical detection/measurement techniques whichrely, at least in part, on the formation of a complex between thechemical species being assayed (the “analyte”) and another species towhich it will bind specifically (a “specific binding partner”). Suchmethods typically employ a labeled ligand analog of the target analyte,the ligand analog selected so that it binds competitively with theanalyte to the specific binding partner. The ligand analog is labeled sothat the extent of binding of the labeled ligand analog with thespecific binding partner can be measured and correlated with thepresence and/or concentration of the target analyte in the biologicalsample.

Numerous labels have been employed in such affinity based sampleanalysis techniques, including enzyme labeling, radioisotopic labeling,fluorescent labeling, and labeling with chemical species subject toelectrochemical oxidation and/or reduction. The use of redox reversiblespecies, sometimes referred to as electron transfer agents or electronmediators as labels for ligand analogs, have proven to provide apractical and dependable results in affinity-based electrochemicalassays. However, the use of electrochemical techniques in detecting andquantifying concentrations of such redox reversible species (correlatingwith analyte concentrations) is not without problem. Electrochemicalmeasurements are subject to many influences that affect the accuracy ofthe measurements, including not only those relating to variations in theelectrode structure itself and/or matrix effects deriving fromvariability in liquid samples, but as well those deriving frominterference between multiple electroactive species, especially whenassay protocols require detection or quantification of multipleelectroactive species.

The present invention relates to novel diffusible, redox-reversibleosmium-bipyridyl conjugates useful in immunosensors based on eitherindirect amplified electrochemical detection techniques or on directelectrochemical measurement of detectable species with microarrayelectrodes under bipotentiostatic control. An Os-bipyridyl complex can,for example be covalently attached to a peptide which has amino acidsequence of the binding epitope for an antibody. When Os complex/peptideconjugate is bound to antibody, the conjugate does not functionelectrochemically; it is said to be “inhibited”. Typically an analytepresent in sample will compete with Os-bipyridyl complex/peptideconjugate for the limited number of binding sites on the antibody. Whenmore analyte is present, more free Os-bipyridyl complex/peptideconjugate will be left in an unbound diffusible state producing highercurrent at a sensor electrode, i.e. one of the working electrodes wheremeasured events (oxidation or reduction) are taking place. In theopposite case, when less analyte is present, more indicator/peptideconjugate will be bound to antibody resulting less free conjugates andproducing lower current levels at the working electrodes. Therefore thecurrent detected at either one of the working electrodes will be afunction of analyte concentration.

It is frequently desired to measure more than one analyte species in aliquid sample. Measurement of multiple species in a mixture has beenachieved with photometry and fluorescence, via selection of theappropriate wavelengths. Electrochemical measurements of a singlespecies in a complex mixture are routinely made by selecting a potentialat which only the desired species is oxidized or reduced (amperometry)or by stepping or varying the potential over a range in which only thedesired species changes its electrochemical properties (AC and pulsemethods). These methods suffer from disadvantages including lack ofsensitivity and lack of specificity, interference by charging and matrixpolarization currents (pulse methods) and electrode fouling due to theinability to apply an adequate over potential. Moreover, electrochemicalmeasurements are complicated by interference between the multiplicity ofelectroactive species commonly extant in biological samples.

Electrode structures which generate steady state current via diffusionalfeedback, including interdigitated array electrodes (IDAs) (FIGS. 1 and2) and parallel plate arrangements with bipotentiostatic control areknown. They have been used to measure reversible species based on thesteady state current achieved by cycling of the reversible species. Areversible mediator (redox reversible species) is alternately oxidizedand reduced on the interdigitated electrode fingers. The steady statecurrent is proportionate to mediator concentration (FIG. 3) and limitedby mediator diffusion. A steady state current is achieved within secondsof applying the predetermined anodic (more positive) and cathodic (lesspositive or negative) potentials (FIG. 6) to the microelectrode array.The slope of a plot of the IDA current vs. mediator concentration isdependent on IDA dimensions, and the slope increases with narrowerelectrode spacings (FIG. 7).

The present invention provides novel osmium-bipyridyl complex conjugatesuseful in a method for measuring multiple analyte species in the samesample, and optimally on the same electrode structure, thus improvingthe accuracy of the relative measurements. The present conjugates can beused with other electroactive conjugate species having unique redoxpotentials to provide an electrochemical biosensor with capacity toprovide improved accuracy. Analyte concentration can bemeasured/calculated from electrometric data obtained on the same liquidsample with the same electrode structure, thereby minimizingperturbations due to variability in sample or electrode structure.

The diffusible osmium conjugates of this invention find use in assaybased on the principle of diffusional recycling, where a diffusibleredox reversible species is alternately oxidized and reduced at nearbyelectrodes (the working electrodes), thereby generating a measurablecurrent. As alternate oxidation and reduction is required formeasurement, only electroactive species which are electrochemicallyreversible at the predetermined redox potential are measured therebyeliminating, or at least reducing, the impact or interference fromnon-reversible electroactive species in the sample or otherreversible-redox species having unique (at least 50 millivoltsdifferent) redox potential. Redox reversible species having differentoxidation potentials can be independently measured in a mixture byselecting and bipotentiostatically controlling the oxidizing andreducing potentials for neighboring electrode pairs so that only thespecies of interest is oxidized at the anode and reduced at the cathode.When the working electrodes are dimensioned to allow diffusionalrecycling of the redox-reversible-species at the selected oxidizing andreducing potentials appropriate for that species, a steady state currentis quickly established through the sample and the electrode structure.The magnitude of the current at the working electrodes where themeasurable oxidative and reductive events are taking place, isproportional to the concentration of the diffusible redox reversiblespecies in the sample. When two or more redox reversible species areutilized, they are selected to have redox potentials differing by atleast 50 millivolts, most preferably at least 200 millivolts, tominimize interference between one species and the other in measurementsof the respective steady state currents. The present osmium complexconjugates have unique redox potentials that allow them to be usedwith/in the presence of other electroactive conjugates without (or withminimal) interference.

The present conjugates can be used in any electrode structure/systemwhich allows for diffusional recycling to achieve steady state currentin response to application of pre-selected complex species-specificanodic and cathodic potentials. Suitable electrode structures includeinterdigitated array microelectrodes and parallel plate electrodesseparated by distances within the diffusion distance of the respectiveredox reversible species. The electrode structures typically include areference electrode (e.g., Ag/AgCl), at least two working electrodes,and optionally an auxiliary electrode for current control. In use, aprogrammable bipotentiostat is placed in electrical communication withthe electrode structure for applying the respective anodic and cathodicpotentials specific for each of the respective redox reversible speciesutilized in the method/biosensor. Several novel osmium complexesincluding those of this invention have been developed for use as labelsfor preparing ligand analog conjugates having potential differencessufficient (at least 50 millivolts) to allow the use of two osmiumcomplexes (as opposed to an osmium complex and a ferrocene or otherredox reversible label) in multiple conjugate based electrochemicalassays.

The present osmium conjugates are useful in a method for measuring theconcentration of one or more analytes in a liquid sample. The methodincludes contacting a portion of the sample with pre-determined amountsof at least a first and second redox reversible species having a redoxpotential differing by at least 50 millivolts from that of each otherspecies. Each respective species comprises a liquid sample diffusibleconjugate of a ligand analog of an analyte in the liquid sample and aredox reversible label. The liquid sample is also contacted with apredetermined amount of at least one specific binding partner for eachanalyte to be measured. The diffusible conjugate is selected so that itis capable of competitive binding with the specific binding partner forsaid analyte. The concentration of the diffusibleredox-reversible-species in the liquid sample is then determinedelectrochemically. The sample is contacted with an electrode structure,including a reference electrode and at least first and second workingelectrodes dimensioned to allow diffusional recycling at least one ofthe diffusible redox-reversible-species in the sample, when apredetermined redox-reversible-species-dependent cathodic potential isapplied to one working electrode and a predeterminedredox-reversible-species-dependent anodic potential is applied to thesecond working electrode. Typically, a first cathodic potential isapplied to the first working electrode and a first anodic potential isapplied to the second working electrode to establish current flowthrough the sample due to diffusional recycling of the firstredox-reversible-species without significant interference from thesecond redox-reversible-species. Current flow through one or more of theelectrodes at the first anodic and cathodic potentials is measured.

Similarly current flow responsive to application of second cathodic andanodic potentials to electrodes in contact with the sample is measuredand correlated with measured current flows for known concentrations ofthe respective redox-reversible-species, said concentrations beingproportionate to the respective analyte concentrations.

The reagent components including the present bipyridyl-osmium conjugatesof the invention and the specific binding partners, can be provided inthe form of a test kit for measuring the targeted analyte(s) in a liquidsample, either as separate reagents or, more preferably, combined as amulti-reagent composition, e.g. combined redox reversible species,combined specific binding partners, or combined redox reversible speciesand specific binding partners. The kit optionally, but preferably,includes an electrode structure dimensioned to allow diffusional redoxrecycling of diffusible redox reversible species in the liquid sample.The electrode structure includes conductors for connecting the structureto a bipotentiostat programmed to applyredox-reversible-species-dependent-anodic and cathodic potentials to theelectrode structure and to sense and measure current flow, typically atone or both of the working electrodes, responsive to such potentials.

This invention is based on the preparation and use of novelelectrochemically detectable osmium complexes and covalent conjugates ofsaid complexes having oxidation potentials differing sufficiently fromother redox-reversible complexes to enable their use together with otherosmium or other metal conjugates. Thus, there are provided novel osmiumlabeled ligand analogs capable of binding to a specific binding partnerof a biologically significant analyte. The electrochemically detectableosmium conjugates comprise a tris(bipyridyl) osmium complexcharacterized by fast mediation kinetics and low redox potential (+150mV vs. Ag/AgCl). Another group of osmium complex labeled,electrochemically detectable conjugates that can be used with thepresent complexes in multi-conjugate assay protocols includebis(bipyridyl) imidazoyl haloosmium complexes, which, like thetris(bipyridyl) osmium complexes are characterized by fast mediationkinetics, but the tris(bipyridyl) complexes have a redox potentialsufficiently different from the bis(pyridyl) imidazolyl chloroosmiumcomplexes to allow their use together in assays utilizing microelectrodearrays for measuring more than one analyte in a single liquid sample byconcentration dependent currents amplified by diffusional redoxrecycling.

The present osmium complex conjugates can be used in combination withanother conjugated redox-reversible-species for the measurement of bothglycosylated hemoglobin and hemoglobin in a lysed blood sample. Oneredox-reversible-species preferably comprises an imidazole-osmiumcomplex covalently linked to a ligand analog of either hemoglobin orglycosylated hemoglobin, and a second redox-reversible-speciescomprising a second redox reversible label covalently bound to a ligandanalog of the other of the two target analytes. The method enablesmeasurement of the concentration of both the glycosylated hemoglobin(HbAlc) and the concentration of either total hemoglobin or that ofunglycosylated hemoglobin (HbA₀) thereby enabling calculation of theresults as a ratio of the two measurements (% HbAlc). It is advantageousto assay both HbAlc and total hemoglobin (or HbA₀) using the sameprinciple in a single sample, particularly because ratioing worksminimize biases due to environmental effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an enlarged plan view of an interdigitated array electrode forreversible mediator measurement.

FIG. 2 is a partial cross-sectional view of the electrode of FIG. 1illustrating the conditions of steady state current limited by diffusionof mediator (M).

FIG. 3 is a graphic presentation of dose response currents for abis-(bipyridyl) imidazolyl chloroosmonium mediator peptide conjugate.

FIG. 4 is a graphic illustration of current flow vs. concentration ofglycosylated hemoglobin (HbAlc) in blood samples using an osmiumconjugate and enzyme amplified DC amperometry.

FIG. 5 is a graphic illustration of the inhibition of current flow dueto free conjugate as a function of antibody concentration (Cn) asmeasured using enzyme amplified DC amperometry [C₁,>C₂,>C₃].

FIG. 6 is a graphic illustration of current flow vs. time using aninterdigitated array electrode.

FIG. 7 is a graphic illustration of the effect of the dimensions of theinterdigitated array electrode structure on current flow as a functionof concentration of an osmium conjugate (Os-DSG-Alc).

FIG. 8 is a graphic illustration of current flow as a function ofapplied potential for a liquid sample containing equimolar (50 μM) of abis-(bipyridyl) imidazolyl chloroosmium complex and a tris(bipyridyl)osmium complex of this invention.

FIG. 9 is a graphic presentation of current flow vs. concentration of aferrocene-biotin conjugate in the presence of varying amounts of anosmium complex conjugate on interdigitated array electrodes withbipotentiostatic control.

FIG. 10 is a graphic illustration of the effect of concentration of anunlabeled conjugate (BSA-Alc) on current flow in a solution containingosmium labeled conjugate (osmium-DSG-Alc)) in the presence of threeseparate Alc-recognizing antibody compositions.

FIG. 11 illustrates the structure of a tris(bipyridyl) osmium labeledconjugate of this invention.

FIGS. 12–14 are similar and each depict the chemical structure of abis(bipyridyl) imidazolyl chloroosmium labeled peptide conjugate.

DETAILED DESCRIPTION OF THE INVENTION

The diffusible redox reversible species of this invention is aliquid-sample-diffusible conjugate of a ligand analog of an analyte anda redox reversible bipyridyl-osmium complex. The term “redox reversible”as used herein refers to a chemical species capable of reversibleoxidation and reduction in a liquid sample. Redox reversible labels arewell-known in the art and include ligand complexes of transition metalions, for example iron (ferrocene and ferrocene derivatives), rutheniumand osmium. The conjugate is prepared by linking the ligand analog tothe label either covalently through difunctional linking agents or bycombination of covalent linkages and art-recognized specific bindingentities (for example, biotin-avidin).

More particularly, the present invention is directed to a redoxreversible osmium complex of the formula

wherein

R and R₁ are the same or different and are 2,2′-bipyridyl,4,4′-disubstituted-2,2′-bipyridyl, 5-5′-disubstituted,-2,2′-bipyridyl,1,10-phenanthrolinyl, 4,7-disubstituted-1,10-phenanthrolinyl, or5,6-disubstituted-1,10-phenanthrolinyl, wherein each substituent is amethyl, ethyl, or phenyl group,

R₅ is 4-substituted-2,2′-bipyridyl or 4,4′-disubstituted-2,2′-bipyridylwherein the 4-substituent is the group B-(L)_(k)-Q(CH₂)_(i)— and the4′-substituent is a methyl, ethyl or phenyl group;

R, R₁ and R₅ are coordinated to Os through their nitrogen atoms;

Q is O, S, or NR₄ wherein R₄ is hydrogen, methyl or ethyl;

—L— is a divalent linker;

k is 1 or 0;

i is 1, 2, 3, 4, 5or 6;

B is hydrogen or a group comprising a ligand capable of binding to aspecific binding partner;

d is +2 or +3;

X and Y are anions selected from monovalent anions, chloride, bromide,iodide, fluoride, tetrafluoroborate, perchlorate, and nitrate anddivalent anions, e.g., sulfate, carbonate or sulfite wherein x and y areindependently 0, 1, 2, or 3 so that the net charge of X_(x)Y_(y) is −2or −3.

Another redox reversible osmium complex that can be used with thepresent imidazole-osmium conjugates is a compound of the formula

wherein

R and R₁ are the same or different and are 2,2′-bipyridyl,4,4′-disubstituted-2,2′-bipyridyl, 5-5′-disubstituted,-2,2′-bipyridyl,1,10-phenanthrolinyl, 4,7-disubstituted-1,10-phenanthrolinyl, or5,6-disubstituted-1,10-phenanthrolinyl, wherein each substituent is amethyl, ethyl, or phenyl group,

R and R₁ are coordinated to Os through their nitrogen atoms;

q is 1 or 0;

R₇ is B-(L)_(k)-Q(CH₂)_(i)—;

R₂ is hydrogen, methyl, or ethyl when q is 1, and R₂ isB-(L)_(k)-Q(CH₂)_(i)—when q is 0; wherein in the groupB-(L)_(k)-Q(CH₂)_(i)-Q is O, S, or NR₄ wherein R₄ is hydrogen, methyl orethyl; —L— is a divalent linker;

-   -   k is 1 or 0;    -   i is 1, 2, 3, 4, 5 or 6; and    -   B is hydrogen or a group comprising a ligand capable of binding        to a specific binding partner;

Z is chloro or bromo;

m is +1 or +2;

X is monovalent anion, e.g., chloride, bromide, iodide, fluoride,tetrafluoroborate, perchlorate, nitrate, or a divalent anion, e.g.,sulfate, carbonate, or sulfite;

Y is monovalent anion, e.g., chloride, bromide, iodide, fluoride,tetrafluoroborate, perchlorate or nitrate; and

n is 1 or zero,

provided that when X is a divalent anion, n is zero,

and when m is 1, n is zero and X is not a divalent anion.

Redox reversible conjugate species of each of those formulas areprepared from the corresponding compounds wherein k is 0 and B ishydrogen by reacting such compounds with either a heterofunctionalcrosslinker of the formula S-L′-T wherein L′ is a divalent linker and Sand T are different electrophilic groups capable of reacting with anucleophilic group to form a covalent bond, or with a homofunctionalcrosslinker of the formula S-L′-T wherein L′ is a divalent linker and Sand T are the same electrophilic groups capable of reacting with anucleophilic group to form a covalent bond. The resulting products arethen reacted with ligand analogs using classical coupling reactionconditions to product the conjugate species. The oxidizing potentials ofthe respective bis(bipyridyl) imidazolyl and tris(bipyridyl) osmiumcomplexes defined above is such that the respective complexes can beused as reversible redox labels for the respective redox reversiblespecies in performance of the method. FIG. 8 illustrates a cyclicvoltammogram for a liquid sample containing equimolar (50 μM) amounts ofa bis(bipyridyl) imidazolyl chloroosmium complex and a tris(bipyridyl)osmium complex.

In one embodiment of the invention the specific binding partner for eachanalyte is an antibody and the ligand analog is selected so that itbinds competitively with the analyte to the antibody. There are,however, other examples of ligand-specific binding partner interactionsthat can be utilized in developing applications of the present method.Examples of ligands and specific binding partners for said ligands arelisted below.

Ligand Specific Binding Partner Antigen (e.g., a drug Specific antibodysubstance) Antibody Antigen Hormone Hormone receptor Hormone receptorHormone Polynucleotide Complementary polynucleotide strand Avidin BiotinBiotin Avidin Protein A Immunoglobulin Immunoglobulin Protein A EnzymeEnzyme cofactor (substrate) Enzyme cofactor (substrate) Enzyme LectinsSpecific carbohydrate Specific carbohydrate Lectins of lectins

The term “antibody” refers to (a) any of the various classes orsubclasses of immunoglobulin, e.g., IgG, IgM, derived from any of theanimals conventionally used, e.g., sheep, rabbits, goats or mice; (b)monoclonal antibodies; (c)intact molecules or “fragments” of antibodies,monoclonal or polyclonal, the fragments being those which contain thebinding region of the antibody, i.e., fragments devoid of the Fc portion(e.g, Fab, Fab′, F(ab′)₂) or the so-called “half-molecule” fragmentsobtained by reductive cleavage of the disulfide bonds connecting theheavy chain components in the intact antibody. The preparation of suchantibodies are well-known in the art.

The term “antigen” used in describing and defining the present inventionincludes both permanently antigenic species (for example, proteins,peptides, bacteria, bacteria fragments, cells, cell fragments andviruses) and haptans which may be rendered antigenic under suitableconditions.

The present osmium labeled conjugates are useful alone or in combinationwith other conjugates in methods for measuring the concentration of oneor more analytes in a liquid sample. One method enables two or moreindependent amperometric measurements of the sample on a singleelectrode structure. The method comprises

contacting a volume of said liquid sample with

-   -   1) predetermined amounts of at least a first and second redox        reversible species, each respective species having a redox        potential differing by at least 50 millivolts from that of each        other species, at least one species comprising a liquid sample        diffusible conjugate of a ligand analog of an analyte in the        liquid sample and a redox reversible label, said conjugate        capable of competitive binding with a specific binding partner        for said analyte, and    -   2) a predetermined amount of at least one specific binding        partner for each analyte to be measured; and    -   electrochemically determining the concentration of each of said        diffusible redox-reversible species in the liquid sample by    -   contacting said sample with an electrode structure including a        reference electrode and at least first and second working        electrodes dimensioned to allow diffusional recycling of the        diffusible redox reversible species in the sample when a        predetermine redox-reversible-species-dependent cathodic        potential is applied to one working electrode and a        predetermined redox-reversible-species-dependent anodic        potential is applied to a second working electrode, said        diffusional recycling of said species being sufficient to        sustain a measurable current through said sample,    -   applying a first cathodic potential to the first working        electrode and a first anodic potential to the second working        electrode, said first cathodic and anodic potentials        corresponding to those respective potentials necessary to        establish current flow through the sample due to diffusional        recycling of the first redox reversible species without        significant interference from said second redox reversible        species,    -   measuring current flow at said first anodic and cathodic        potentials,    -   applying a second cathodic potential to said first or second        working electrode and a second anodic potential to the other        working electrode, said second cathodic and anodic potential        corresponding to those respective potentials necessary to        establish current flow through the sample due to diffusional        recycling of the second redox-reversible-species without        significant interference from the first redox reversible        species,    -   measuring current flow at said second anodic and cathodic        potentials, and    -   correlating the respective measured current flows to that for        known concentrations of the respective diffusible redox        reversible species, said concentrations being proportionate to        the respective analyte concentrations.

That method has very broad applicability but in particular may be usedto assay: drugs, hormones, including peptide hormones (e.g., thyroidstimulating hormone (TSH), luteinizing hormone (LH), folliclestimulating hormone (FSH), insulin and prolactin) or non-peptidehormones (e.g, steroid hormones such as cortisol, estradiol,progesterone and testosterone, or thyroid hormones such as thyroxine(T4) and triiodothyronine), proteins (e.g., human chorionic gonadotropin(hCG), carcino-embryonic antigen (CEA) and alphafetoprotein (AFP)),drugs (e.g., digoxin), sugars, toxins or vitamins.

The method can be performed on liquid samples comprising biologicalfluids such as saliva, urine, or blood, or the liquid sample can bederived from environmental sources. The liquid samples can be analyzed“as is,” or they can be diluted, buffered or otherwise processed tooptimize detection of the targeted analyte(s). Thus, for example, bloodsamples can be lysed and/or otherwise denatured to solubilize cellularcomponents.

The method can be performed using widely variant sampling handlingtechniques. Thus, the sample can be premixed with either or both of thespecific binding partner for the targeted analytes and the redoxreversible species prior to contacting the sample with the electrodestructure, or the liquid sample, either neat or pre-processed, can bedelivered to a vessel containing predetermined amounts of the redoxreversible species and the specific binding partner for subsequent orsimultaneous contact with the electrode structure. The order ofintroduction of the components into the sample is not critical; however,in one embodiment of the invention the predetermined amounts of thespecific binding partners are first added to the sample, and thereafter,there is added the predetermined amounts of the redox reversiblespecies. It is also possible to combine the predetermined amounts of thespecific binding partners with the redox reversible species to form therespective complexes prior to combining those components with the liquidsample. In that latter case the redox reversible species will bedisplaced from its respective specific binding partner by thecorresponding analyte to provide a concentration of the redox reversiblespecies proportionate to the concentration of analyte in the liquidsample. The reagents, that is, the predetermined amounts of the specificbinding partner of each analyte and the predetermined amounts of thecorresponding redox reversible species can, for example, be deposited ina vessel for receiving a predetermined volume of the liquid sample. Theliquid sample is added to the vessel, and thereafter, or simultaneously,the liquid sample is contacted with the electrode structure.

The electrode structure includes a reference electrode and at leastfirst and second working electrodes dimensioned to allow diffusionalrecycling of the diffusible redox reversible species in the sample whenpredetermined redox-reversible-species-dependent-cathodic and anodicpotential is applied to the working electrodes. The term “workingelectrode” as used herein refers to an electrode where measured events(i.e. oxidation and/or reduction) take place and resultant current flowcan be measured as an indicator of analyte concentration. “Anodicpotential” refers to the more positive potential (applied to the anode)and “cathodic potential” refers to the less positive or negativepotential applied to the cathode (vs a reference electrode). Electrodesdimension to allow diffusional recycling are well known in the art andare typically in the form of arrays of microdiscs, microholes, ormicrobands. In one embodiment the electrodes are in the form of aninterdigitated arrangement of microband electrodes with micron orsubmicron spacing. Short average diffusional length and a large numberof electrodes are desirable for effective current amplication byrecycling of reversible redox species. The microelectrode arrays can befabricated, for example, as pairs of interdigitated thin film metalelectrodes in micron and submicron geometry arranged on an insulatorsubstrate, for example, oxidized silicon. Each of the electrode fingers(FIG. 1) are spaced from its neighboring finger in the nanometer to lowmicrometer (1–10 microns) range. Microelectrode arrays can be fabricatedusing photolithography, electron bean lithography, and so-calledlift-off technique. Thus, an interdigitated electrode array (IDA) can bedeposited on glass, silicon or polyamide utilizing the following generalprocedure:

-   -   1. Grow thermal oxide layer on silicon substrate;    -   2. Sputter 400 Å chromium seed layer, 2000 Å gold;    -   3. Spin-coat and soft-bake photo resist;    -   4. Expose and develop photo resist with IDA pattern;    -   5. Pattern gold and chromium with ion beam milling;    -   6. Strip photo resist; and    -   7. Cut electrodes into chips by first coating with a protective        layer, cutting into strips, stripping the protective layer, and        cleaning electrode surfaces in oxygen plasma.

The electrode structure can be formed on an inner surface of a chamberfor receiving the liquid sample, e.g., a cuvette, a capillary fillchamber, or other sample receiving vessel wherein the electrodestructure can be contacted with the liquid sample. Alternatively, theelectrode structure can form part of a probe for dipping into the liquidsample after the sample has been contacted with the predeterminedamounts of the redox reversible species and the specific bindingpartners. The electrode structure is in contact with conductors thatenable application of the respective cathodic and anodic potentials forcarrying out the present method. The anodic and cathodic potentials areapplied relative to a reference electrode component of the electrodestructure using a bipotentiostat. The electrode structure can optionallyinclude an auxiliary electrode for current control. The bipotentiostatis utilized to apply a first cathodic potential to a first workingelectrode and a first anodic potential to a second working electrode,the first cathodic and anodic potentials corresponding to thoserespective potentials necessary to establish current flow through thesample due to diffusional recycling of the first redox reversiblespecies. Optionally the potential on one working electrode can be set ata first diffusible species dependent, anodic potential and current flowis measured as the potential of the other working electrode is sweptthrough a potential corresponding to the predetermined diffusiblespecies dependent cathodic potential (or vice versa).

The cathodic and anodic potentials appropriate for each reversible redoxspecies can be readily determined by empirical measurement. The multipleredox reversible species used in performance of the method of thisinvention are selected to have redox potentials differing by at least 50millivolts, more preferably at least 100 millivolts, more preferably atleast 200 millivolts, from that of each other redox reversible speciesutilized in the method. The difference in redox potentials of the redoxreversible species being used allow each species to be detected withoutsignificant interference from the second or any other redox reversiblespecies in the liquid sample. A steady state current flow is rapidlyestablished at each of the working electrodes following application ofthe anodic and cathodic potentials. Current flow can be measured ateither or both working electrodes, and it is proportionate to theconcentration of the recycling redox reversible species.

Second cathodic and anodic potentials are applied to the workingelectrodes wherein said second potentials correspond to those respectivepotentials necessary to establish current flow through the sample due todiffusional recycling of the second redox reversible species withoutsignificant interference from the first redox reversible species, andthe resulting steady state current flow is measured. This step isrepeated for each redox reversible species utilized in the method. Themeasured current flows are then correlated to known concentrations ofthe respective diffusible redox reversible species. Those concentrationsare proportionate to the respective analyte concentrations.

The method steps can be conducted using a programed bipotentiostat tocontrol potentials on the electrode structure in contact with thesample. The bipotentiostat can be included either in a desktop orhand-held meter further including means for reading values for steadystate current, storing said values, and calculating analyteconcentrations using a microprocessor programmed for making suchcalculations.

The relative amounts of the first and second redox reversible speciesand the respective specific binding partners for the targeted analytesto be measured in the method can be determined empirically. They aredependent on the concentration ranges of the targeted analyte, and thebinding stoichiometry of the specific binding partner, the bindingconstant, the analyte and the corresponding redox reversible species.The amounts of each reagent appropriate for each analyte being measuredcan be determined by empirical methods.

The present osmium conjugates can also be used in a method for measuringtwo proteinaceous analytes in a liquid sample wherein the ligand analogcomponent of the first redox reversible species is a peptide comprisingan epitope of a first analyte and the ligand analog component of asecond redox reversible species is a peptide comprising an epitope of asecond analyte. One specific binding partner utilized in the method isan antibody recognizing the epitope of the first analyte, and the otherspecific binding partner is an antibody recognizing the epitope of thesecond analyte. In another application of that method two independentmeasurements are performed on a single analyte in a liquid sample. Inthat embodiment the respective ligand analog component of the first andsecond redox reversible species are different ligand analogs of thetargeted analyte. Where the targeted analyte is a proteinaceouscompound, the ligand analog component of the first redox reversiblespecies is a peptide comprising a first epitope of the analyte, and theligand analog of the second redox reversible species is a peptidecomprising a second epitope of the analyte, and the specific bindingpartners are first and second antibodies, each recognizing respectivefirst and second analyte epitopes.

The present osmium conjugates can also be used in a device for detectingor quantifying one or more analytes in a liquid sample. The devicecomprises

at least two redox reversible species, each capable of diffusion in saidliquid sample at least in the presence of a respective predeterminedanalyte, said redox reversible species having respective redoxpotentials differing by at least 50 millivolts,

an electrode structure for contact with the liquid sample in saidchamber, said electrode structure including a reference electrode andworking electrodes dimensioned to allow diffusional recycling of adiffusible redox reversible species in a liquid sample in contact withthe electrode system when a predeterminedredox-reversible-species-dependent cathodic potential is applied to oneworking electrode and a predetermined redox-reversible-species-dependentanodic potential is applied to a second working electrode, saiddiffusional recycling of said species being sufficient to sustainmeasurable current through each working electrode, and

conductors communicating with the respective electrodes for applyingsaid anodic potential and said cathodic potential and for carrying thecurrent conducted by the electrodes.

The device can be constructed using procedures and techniques that havebeen previously described in the art for construction of biosensorsemploying electrometric detection techniques. Thus, for example, thedevice can include a chamber that has a receiving port, and the chamberis dimensioned so that it fills by capillary flow when the liquid sampleis contacted with the sample receiving port. The electrode structure canbe formed on a plate that defines a wall of the chamber so that theelectrode structure will contact a liquid sample in the chamber. Thus,for example, the device can be constructed using the general proceduresand designs described in U.S. Pat. No. 5,141,868, the disclosure ofwhich is expressly incorporated herein by reference. The features of thepresent invention can also be incorporated into other electrochemicalbiosensors or test strips, such as those disclosed in U.S. Pat. Nos.5,120,420; 5,437,999; 5,192,415; 5,264,103; and 5,575,895, thedisclosures of which U.S. patents are expressly incorporated herein byreference. The device can be constructed to include the predeterminedamounts of the redox reversible species and the specific bindingpartners. For example, a mixture of such reagents can be coated onto awall of the sample chamber in said device during device construction, sothat the liquid sample is contacted with the reagent mixture as it isdelivered into the chamber for containing the sample. In one embodimentthe device is constructed for quantifying a first analyte and a secondanalyte in liquid sample. The device comprises two redox reversiblespecies, a first redox reversible species comprising a conjugate of aligand analog of the first analyte and a second redox reversible speciescomprising a conjugate of a ligand analog of the second analyte, and aspecific binding partner for each analyte so that each of said analyteanalog conjugates are capable of binding competitively with itsrespective analyte to a specific binding partner.

In another application of the present osmium conjugates, they are usedin conjunction with a device that further comprises a bipotentiostat inelectrical communication with the conductors for applying a redox-reversible-species-dependent-cathodic potential to one working electrodeand a redox-reversible-species-dependent-anodic potential to a secondworking electrode. The biopotentiostat can be programmed to apply asequence of potentials to the respective working electrodes. Moreparticularly, the bipotentiostat can be programmed to apply firstcathodic potential to a first working electrode and a first anodicpotential to a second working electrode, said first anodic and cathodicpotentials corresponding to those potentials necessary to establishcurrent flow to the sample due to diffusional recycling of the firstredox reversible species. The bipotentiostat is also programmed to applya second cathodic potential to said first working electrode and a secondpotential to the second anodic electrode, said second cathodic andanodic potentials corresponding to those potentials necessary toestablish current flow through the sample due to diffusional recyclingof the second redox reversible species. In an alternate embodiment thedevice includes first and second redox reversible species, and at leastfirst and second electrode structures for contact with the liquid samplein the chamber, each of said electrode structures comprising amicroarray of working electrodes, and means for switching thebipotentiostat between the first and second electrode structures. Inpreferred device embodiments there is provided means for measuringcurrent flow through the sample at each of the first and secondpotentials and preferably storing values for said current flows in aregister coupled to a microprocessor programmed to calculate analyteconcentrations based on said values.

In still another embodiment the present conjugate is used in a kit formeasuring the concentration of one or more analytes in liquid sample.The kit comprises

at least two redox reversible species for contact with the liquidsample, each capable of diffusion in the liquid sample at least in thepresence of a predetermined analyte, at least one of such species beinga conjugate of a ligand analog of an analyte and a redox reversiblelabel, said redox reversible species having respective redox potentialsdiffering by at least 50 millivolts;

a specific binding partner for each analyte;

an electrode structure for contact with the liquid sample, saidelectrode structure including a reference electrode and workingelectrodes dimensioned to allow diffusional recycling of diffusibleredox reversible species in the sample when a predeterminedredox-reversible-species-dependent-cathodic potential is applied to oneworking electrode and a predeterminedredox-reversible-species-dependent-anodic potential is applied to thesecond working electrode, said diffusional recycling of said speciesmeans sufficient to sustain a measurable current through the sample; and

conductors communicating with the respective electrodes for applyingsaid anodic potential and said cathodic potential and for carrying thecurrent conducted by the electrodes.

In one embodiment, the present redox reversible conjugate species aremixed with other electroactive species as a novel composition forcontact with the liquid sample. In another embodiment each of the redoxreversible species and the specific binding partner for each analyte ismixed as a novel composition for contact with the liquid sample.Preferably, the redox reversible label of at least one of the redoxreversible species comprises an osmium complex of this invention.

Preparation of Os Mediator Labels

The Os mediator bis(bipyridyl) imidazolyl chloroosmium has been shown tobe an excellent electron mediator for many oxide-reductase enzymes (U.S.Pat. No. 5,589,326). It has fast mediation kinetics (about 500 timesfaster than ferricyanide with glucose oxidase) and a relatively lowredox potential (+150 mV vs. Ag/AgCl). It has also very fast electrontransfer rate at electrode surface. More importantly, the organicligands on Os mediator can be functionalized so that it can becovalently linked to other molecules without detrimental effects onredox properties of the Os center. These unique properties of Osmediator make it an ideal electrochemical indicator for sensors based onimmunoaffinity.

Os mediators with these new ligands were synthesized using the sameprocedure used for Os free mediator. Their synthesis consists of twomajor process steps as outlined below. Details of these processing stepsare described below.

The first process step involves the synthesis of Os intermediate,cis-bis(2,2′-bipyridyl)dichloroosmium(II), from commercially availableosmium salt using the following scheme. The intermediate product isisolated through recrystallization in an ice bath.

${K_{2}{Os}^{IV}{Cl}_{6}} + {2\;{bpy}{\frac{DMF}{\Lambda}\left\lbrack {{{Os}^{III}({bpy})}_{2}{Cl}_{2}} \right\rbrack}{Cl}} + {2{KCl}}$${{2\left\lbrack {{{Os}^{III}({bpy})}_{2}{Cl}_{2}} \right\rbrack}{Cl}} + {{Na}_{2}S_{2}O_{4}} + {2H_{2}{O\mspace{14mu}}^{\underset{\_}{0{^\circ}\mspace{14mu}{C.}}}}$2Os^(II)(bpy)₂Cl₂↓+2Na⁺+2SO₃ ⁼+4H⁺+2Cl

The second process step involves the reaction between Os intermediateand histamine or 4-imidazoleacetic acid (or a substituted bipyridine forpreparation of the tris(bipyridyl) complexes) to produce Os mediatorswith the appropriate “handle”. The desired product is then precipitatedout from solution by addition of ammonium tetrafluoroborate.

${{{Os}^{II}({bpy})}_{2}{Cl}_{2}} + {{histamine}{\frac{{{EtOH}/H_{2}}O}{\Delta}\left\lbrack {{{Os}^{II}({bpy})}_{2}({histamine})} \right\rbrack}{Cl}}$[Os^(II)(bpy)₂(histamine)Cl]Cl+NH₄BF₄→[Os^(II)(bpy)₂(histamine)Cl]BF₄↓+NH₄Cl

These Os mediators can also be easily converted to oxidized form, i.e.Os(III) using nitrosonium tetrafluoroborate. However, this isunnecessary since the Os revert back to reduced form anyway at alkalineconditions during conjugation reactions. And it does not requireoxidized form of Os(III) for the detection on the biosensor.

A. Simple Mixed Mediator Measurement

1. Interdigitated array microelectrodes (IDA) are produced throughphotolithographic means by art-recognized methods, (See WO 97/34140; EP0299,780; D. G. Sanderson and L B. Anderson, Analytical Chemistry, 57(1985), 2388; Koichi Aoki et al., J Electroanalytical Chemistry, 25691988) 269; and D. Niwa et la., J Electroanalytical Chemistry, 167(1989) 291. Other means which are standard in lithographic processingmay also be used to produce the desired patterns of a conductor oninsulator substrate.

2. Reversible mediators are selected from those described herein andthose described references (U.S. Pat. Nos.4,945,045 and 5,589,325, thedisclosures of which are incorporated herein by reference). Preferablytwo different mediators are selected with potentials which differ by atleast 100 mV, more preferably at least 200 mV. Examples of suitablemediators include the Os(bipy)₂ImCl of this invention and in U.S. Pat.No. 5,589,326, the disclosure of which is incorporated herein byreference, and ferrocene, described in U.S. Pat. No. 4,945,045 and EP0142301, the disclosures of which are incorporated herein by reference.Mixtures of these mediators are made in aqueous solution, for examplephosphate-buffered saline (PBS). Concentrations between about 1 uM and1000 uM may conveniently be measured.

3. The IDA is connected to a bipotentiostat, an instrument capable ofcontrolling the potential of two separate electrodes. Also provided is areference electrode. This non-polarizable electrode serves as thereference for the two applied potentials and may also serve as thecounter electrode. Any non-polarizable electrode may be used, forexample Ag/AgCl, such as may be obtained from ABI/Abtech. An auxiliaryelectrode can also be used for controlling current flow through theworking electrodes. The mixtures are placed on the IDA electrode and thereference electrode also contacted with the mixture, or the IDA alongwith the reference electrode may be dipped into the mixture.

4. To measure Mediator 1 (Os(bipy)₂ImCl)

A cathodic potential is applied to one set of fingers of the IDA whichis capable of reducing mediator 1 (ca −50 mV vs. Ag/AgCl). An anodicpotential is applied to the other set of fingers of the IDA which iscapable of oxidizing mediator 1 but not mediator 2 (or any othermediators) (ca 250 mV vs Ag/AgCl). After a short time (msec to sec), asteady state current will be measurable which is dependent only on theconcentration of mediator 1.

5. To measure Mediator 2 (Ferrocene)

A cathodic potential is applied to one set of fingers of the IDA whichis capable of reducing mediator 2 but not mediator 1 (ca 250 mV vs.Ag/AgCl). An anodic potential is applied to the other set of fingers ofthe IDA which is capable of oxidizing mediator 2 (ca 550 mV vs Ag/AgCl).After a short time (msec to sec), a steady state current will bemeasurable which is dependent only on the concentration of mediator 2.

Specific Binding Assay with Mixed Mediator Measurement.

Specific Assay of HbAlc in a blood sample

1. IDA electrodes are provided as in Paragraph A above.

2. Conjugates of mediators 1 and 2 and haptens or specific bindingmembers are provided using art-recognized procedures for covalentcoupling using either a homo-functional or hetero-functional linker.Specifically, a synthetic peptide corresponding to the N-terminalsequence of the β-chain of HbAlc is conjugated to the osmium complex.Similarly, a synthetic peptide corresponding to the N-terminal sequenceof HbA0 is conjugated to a second mediator, for example ferrocene.

3. Antibodies for the analytes (HbAlc and HbA0) which react specificallywith the N-terminal peptides which have been incorporated into theconjugate are provided by standard methods for producing polyclonalantibodies. In this case, sheep were immunized with carrier proteins towhich were conjugated the synthetic peptide sequences for HbAlc andHbA0. Following the appropriate immunization schedule, the sheep werebled, and the antibody isolated from the blood via ion exchangechromatography, followed by immunosorbent purification on a column ofthe same N-terminal peptide with a different linker.

4. Appropriate stoichiometry of the reaction was determined for the tworeactions independently by methods standard for immunoassay development.A solution containing a fixed amount of labeled conjugate was mixed witha solution with varying amounts of antibody, and, following anappropriate incubation period, the amount of free conjugate remainingwas measured on the IDA electrode using the procedure described above.The amount of antibody just sufficient to achieve maximum inhibition ofthe conjugate (ca>80%) was selected.

5. Reagent solution 1 was made containing a mixture of the twoconjugates in the appropriate concentrations. Reagent solution 2 wasmade containing a mixture of the two antibodies in the amountsdetermined above. A blood sample was diluted ca 20-fold in a solution of25 mM citric acid/0.5% Brij-35. Following a 30 second incubation toallow for lysis and denaturation of the hemoglobin, to 66 uL of thisdiluted sample was added 33 uL of 1 M phosphate buffer, to adjust the pHback to neutral. 30 uL of antibody solution 2 was added, and the mixtureallowed to incubate 30 sec. Then 30 uL of conjugate solution 1 wasadded, and the mixture measured on the IDA electrode. The concentrationof HbAlc in the sample is related to the current measured from Mediator1, and the concentration of HbA0 is related to the current from Mediator2. The %HbAlc in the sample is related to the ratio of the measuredamounts of Mediator 1 and Mediator 2.

Application to HbAlc Assay

Hemoglobin Alc is a specific glycohemoglobin in which the glycosylationtakes place at the N-terminal of hemoglobin b-chain. The antibody bindsspecifically to HbAlc has an epitope sequence of Gluc-Val-His-Leu-Thr(SEQ ID NO:1). To facilitate conjugation to other molecules, a nonnativeamino acid has been added to the sequence, e.g., Cys, Lys, or Lys-MH, toproduce Alc peptides including: 1) Gluc-Val-His-Leu-Thr-Lys-MH (SEQ IDNO:2); 2) Gluc-Val-His-Leu-Tbr-Lys (SEQ ID NO:3); 2)Gluc-Val-His-Leu-Thr-Cys (SEQ ID NO:4).

HbAlc assay requires measuring both Alc concentration ahd totalhemoglobin concentration and reports the results as a ratio of these twomeasurements (%HbAlc). It is advantageous to assay both Alc and totalhemoglobin using same principle because ratioing would minimize biasesdue to environmental effects. Thus antibody has been raised to bindspecifically to hemoglobins with unglycosylated N-terminus, i.e. with anepitope sequence of Val-His-Leu-Tbr (SEQ ID NO:5). Similarly, nonnativeamino acid is added to the sequence to facilitate conjugation. Thepeptides used for total hemoglobin measurement is termed as A0 peptide.A0 peptides that have been used in the preparation of Osmediator-peptide conjugates include Val-His-Leu-Thr-Cys (SEQ ID NO:6)and Val-His-Leu-Thr-Lys (SEQ ID NO:7).

Conjugation Chemistry and Conjugates

There are many types of conjugation chemistry that can be employed tolink Os mediator to a peptide. The following two conjugation chemistriesemployed for the preparation of Os mediator-peptide conjugates have alsobeen commonly used for preparing protein conjugates: 1) formation ofamide bond by reactive ester with primary amine; 2) formation ofthioether bond by maleimide with sulfhydryl group. Amide bond ispreferred over thioether bond because amide bond is generally morestable. Based the preferred conjugation chemistry, the ligand on Osmediator can be functionalized with either a primary amine group or acarboxylic acid group. The best location for these functional groups isbelieved to be the C-4 or C-5 positions on the imidazole ligand of Osmediator, however, functionalization through the non-Os-complexedimidazole ring nitrogen atom can also be carried out. Two differentfunctionalized Os mediators were synthesized as described above.

Os mediator (a) was formed with histamine while Os mediator (b) wasformed with imidazolacetic acid. However, it was found that the iminonitrogen of the imidazole ring interferes with the activation ofcarboxylic acid group to reactive ester (i.e., N-hydroxysuccinimideester) using carbodiimide. Thus, use of carboxylic acid functionalizedOs mediator in the synthesis of Os mediator-peptide conjugates gave muchless favorable results.

The amine group on histamine ligand of Os mediator readily reacts withN-hydroxysuccinimide (NHS) ester to form amide bond. Two types ofcrosslinkers have been employed to link Os mediator to peptides, (a)heterofunctional crosslinker, having a NHS ester at one end and theother end has a maleimide or a sulfhydryl group; and (b) homofunctionalcrosslinker, e.g. both ends have NHS esters.

In the case of heterofunctional crosslinker, the crosslinker is firstreacted with Os mediator with histamine ligand (Os histamine) at 1:1molar ratio. One particular point needs to be noted here. Os mediator inoxidized form, i.e. Os(III), can instantly oxidize sulfhydryl group toform disulfide bond. It is important to keep Os center in the reducedform by addition of a small amount of reductant such as sodiumdithionite during the conjugation processes. The reaction progress canbe monitored by analytical reverse-phase HPLC on a C18 column. Then theOs mediator-crosslinker adduct is isolated via preparative HPLC and thecollected fraction is subsequently freeze-dried. Finally, the Osmediator-crosslinker adduct is reacted with the appropriate peptide toform Os mediator-peptide conjugate. Again, the product is isolated bycollecting appropriate fraction in preparative HPLC and the collectedfraction is then freeze-dried.

Two different heterofunctional crosslinkers have been used for thesynthesis of Os mediator-peptide conjugates. SMCC (succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate) is used forcystein-containing peptide, while SATA (N-succinimidylS-acetylthioacetate) is used for maleimide-containing peptide. Three Osmediator-peptide conjugates (two with Alc peptide and one with A0peptide) have been made using heterofunctional crosslinkers and theirstructure are shown below: (a) Os-SMMC-Alc; (b) Os-SATA-Alc, and (c)Os-SATA-AO.

However, it has been found that these conjugates were not stable whenthey were stored as solutions. Analytical HPLC results indicated thatthese conjugates degrade. Mass spectroscopy confirmed that theinstability is due to splitting of thioether bond present in theseconjugates.

In order to avoid thioether bond in the conjugate, homofunctionalcrosslinker containing two NHS esters was used instead to prepare theconjugates. The crosslinker used was DSG (disuccinimidyl glutarate). Inorder to prevent the formation of crosslinked Os mediator, i.e.Os-crosslinker-Os, a large excess of homofunctional crosslinker was usedin the reaction with Os histamine at 4:1 molar ratio. Under thiscondition, only the desired product, i.e. Os-crosslinker, was formed.The Os-DSG-Alc conjugate was similarly prepared using the proceduredescribed earlier.

The preparation of analogous Os-DSG-A₀ conjugate requires and extra stepsince the unglycated N-terminal amine of HbA₀ peptide is also reactivetoward NHS ester. In this case, the N-terminal amine of HbA₀ peptide isfirst protected by either a base-labile Fmoc¹ or an acid-labile Boc²group. After reacting with Os-DSG adduct to form Os-DSG-A₀ conjugate,the protecting group is then cleaved using appropriate deprotectionmethod (adding base for Fmoc or acid for Boc). The peptides prepared bysolid-phase peptide synthesis already have N-terminal Fmoc protectinggroups. The protecting groups are usually removed prior to cleavage ofpeptide from the resin beads, but they can also be left on if sodesired. The HbA₀ peptide from Zymed has an intact Fmoc protecting groupat N-terminal. Using this strategy the Os-DSG-A₀ conjugate wassuccessfully synthesized.¹Fmoc=9-flourenylmethyloxycarbonyl²Boc=t-butyloxycarbonyl

Many analytes cannot be assayed using enzyme-based sensors. They requirethe development of affinity biosensors or immunosensors which are basedon the selective binding of antigens to antibodies. The key to thedetection of this binding event on electrochemical sensors is theinclusion of antigens labeled with redox labels. Bis(2,2′-bipyridyl)imidazolyl chloroosmium, a.k.a. Os mediator, possesses many propertiesthat make it an excellent redox label for this purpose. In addition, a“handle” for linking it covalently to antigens can be added on withoutaffecting its redox properties.

Several assay schemes can be used in affinity biosensors including, i)competitive binding assay (labeled antigen is competing for a limitednumber of binding sites); ii) sequential binding assays (labeled antigenis bound to excess binding sites); iii) heterogeneous assay (uses aseparation step to separate bound and free labeled antigens); and iv)homogeneous assay (no separation step). The steps involved in ahomogeneous sequential binding assay include binding the analyte to anantibody. The labeled antigen (analyte analog) binds to the remainingbinding sites on the antibody. Finally the leftover free labeled antigenis detected at electrode surface. The resulting current will be afunction of the amount of analyte present.

The detection of free labeled antigens can be achieved using eitherdirect detection or amplified detection methods. Direct detectionrequires the use of advanced electrochemical techniques such as acvoltammetry, differential pulse voltammetry, square wave voltammetry orac impedance in order to reach a sensitivity of 5 μM or less. Amplifieddetection methods use dc amperometry with amplification through reactionwith enzyme or chemical reductants or by using interdigital array (IDA)microelectrodes. The preferred detection method is amplified amperometrythrough cycling of free Os mediator label by using IDA microelectrodes.However, amplified amperometry using Gluc-DOR enzyme can also be used.Fast mediation kinetics of Os mediator is very desirable because themagnitude of amplification is dependent on its mediation kineticconstant with the enzyme.

General Analytical HPLC Method for Osmium Conjugates

All HPLC analysis were performed using a Beckman System Gold HPLC systemconsists of a 126 pump module and a 168 diod array detector module.Stationary phase is a Vydac analytical reverse-phase C18 analyticalcolumn. Other parameters are listed below.

Mobil Phase: A = 0.1% TFA³ in H₂O B = 0.1% TFA in CH₃CN Flow rate: 1mL/min Gradient: 0–5 min: 10% B 5–45 min: 10% B −> 50% B at 1%/min 45–50min: 100% B Detector: Channel A at 384 nm Channel B at 220 nm.Synthesis of Bis(2,2′-bipyridyl)dichloroosmium

-   -   1. Charge a 1 L one-neck RB flask with 19.335 grams K₂OsCl₆        (0.04019 mole) and 13.295 grams 2,2′-dipyridyl (0.08512 mole).        Add 400 mL DMF to dissolve all reactants.    -   2. Heat the solution to reflux and then maintain reflux for 1        hour. Then turn off the heat and let solution cool to 30–40° C.        at ambient.    -   3. Filter the reaction mixture using a medium grade glass-frit        filter. Rinse the flask with additional 20 mL DMF and wash the        filter.    -   4. Transfer the filtrate into a 3-L beaker. Charge another 2-L        beaker with 22.799 grams of NaS204 and dissolve in 2 L deionized        water. Add this solution to the beaker containing Os/DMF        filtrate dropwise using a dropping funnel. Keep the solution        stirring at all time.    -   5. Then cool the mixture in an ice bath for at least 3 hours.        Add ice as necessary.    -   6. Filter the mixture “cold” using a ceramic filter with filter        paper. Wash the content on the filter with 50 mL, water twice        and 50 mL ether twice.    -   7. Dry the product under high vacuum at 50° C. overnight (at        least 15 hours). Weigh the product and transfer into a brown        bottle. Store in a desiccator at room temperature.        Typical yield=16 gram or 70%.        Product is analyzed by UV-Visible spectroscopy and elemental        analysis.

UV-Vis: Peak λ (nm) ε (M⁻¹cm⁻¹) 382 10,745 465  9,914 558 11,560 836 3,873 EA: C % H % N % C1 % Os % H₂O % Theoretical 41.89 2.81 9.77 12.3633.17 0 Actual 40.74 2.92 9.87 11.91 0.41Synthesis of Bis(2,2′-bipyridyl)histamine chloroosmium

-   -   1. Charge a 2 L one-neck RB flask with 11.3959 gram Os(bpy)₂Cl₂        (0.0198 mole) and 4.9836 gram histamine (0.0448 mole). Add 500        mL ethanol to dissolve the reactants. Then add 250 mL deionized        water.    -   2. Heat the solution to reflux and maintain reflux for 6 hours.        Let solution cool to RT at ambient.    -   3. Remove all ethanol using rotary evaporation. Then transfer        the solution into a 500 mL beaker. Dissolve 2.136 gram NH₄BF₄ in        20 mL water. Add dropwise to Os solution. Precipitate forms.        Cool in an ice bath for 30 min. Filter the mixture using a        ceramic filter with filter paper. Wash the content on filter        with ˜20 mL water twice.    -   4. Dry under high vacuum at 50° C. overnight (at least 15 hour).    -   5. Weigh the product and transfer to a brown bottle. Store in a        desiccator at room temperature.        Typical yield=7.6 gram or 52%.        Product is analyzed by UV-visible spectroscopy and HPLC.

UV-Vis: Peak λ (nm) ε (M⁻¹cm⁻¹) 355 7,538 424 7,334 514 7,334 722 2,775HPLC: Elution time = 18.0 min Purity by HPLC range from 65–85%Preparation of [Os(bpy)₂(histamine)Cl]-heterofunctional CrosslinkerAdduct

-   -   1. Weigh 0.1167 g [Os(bpy)₂(histamine)Cl]BF₄ (0.162 mmol) and        transfer to a 5 mL Reacti-Vial. Add 1.0 mL DMF to dissolve the        reactant. Add 25 μL triethylamine.    -   2. Add 0.0508 g SMCC(0.150 mmol)or 0.0390 g SATA (0.168 mmol).        Stir the reactants at RT for 2 hours. Inject a sample into HPLC        to monitor reaction progress.    -   3. If reaction is complete, dilute the solution with 0.1% TFA        buffer to a final volume of 4.5 mL. Inject into preparative HPLC        and collect the product peak.    -   4. Freeze dry the collected fraction overnight.    -   5. Weigh the product and transfer to a brown bottle. Store in a        desiccated bag at −20° C.        Typical yield=40 mg or 25%.        Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-SMCC: Elution time = 32.1 min m⁺/e = 434.2 Os-SATA:Elution time = 27.5 min m⁺/e = 382.8Preparation of [Os(bpy)₂(histamine)Cl]-homofunctional Crosslinker Adduct

-   -   1. Weigh 0.2042 g DSG (0.626 mmol) ) and transfer to a 5 mL        Reacti-Vial. Add 0.75 mL DMF to dissolve the reactant.    -   2. Weigh 0.1023 g [Os(bpy)₂(histamine)Cl]BF₄ (0.142 mmol) and        transfer to a separate 5 mL Reacti-Vial. Add 1.0 mL DMF to        dissolve the reactant. Add 25 μL triethylamine. Then add Os/DMF        solution dropwise to DSG/DW solution with constant stirring.        After reacting for 2 hours at RT, inject a sample into HPLC to        monitor reaction progress.    -   3. If reaction is complete, dilute the solution with 0.1% TFA        buffer to a final volume of 4.5 mL. Inject into preparative HPLC        and collect the product peak.    -   4. Freeze dry the collected fraction overnight.    -   5. Weigh the product and transfer to a brown bottle. Store in a        desiccated bag at −20° C.        Typical yield=45 mg or 35%.        Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-DSG: Elution time = 27.1 min m⁺/e = 429.2 and 859.6Preparation of Os-SATA-Alc Conjugate

-   -   1. Weigh 40.5 mg Os-SATA (0.0529 mmol) and transfer to a 5 mL        Reacti-Vial with stir bar. Add 1.0 mL PBS (pH 7.5) to dissolve.        Add 20 mg Na₂S₂O₄ in order to keep Os in reduced form.    -   2. Add 1.0 mL deacetylation buffer (PBS pH7.5+0.5 M        hydroxylamine and 25 mM EDTA) to deprotect the sulfhydryl group.        Inject a sample into analytical HPLC to determine whether        deprotection is complete by appearance of a new peak at 25.8        min.    -   3. Add 45 mg HbAlc-MH peptide (0.0474 mmol) and let react at RT        for 1 hour. Inject a sample into analytical HPLC to monitor        reaction progress.    -   4. If reaction is complete, dilute the mixture with 0.1% TFA        buffer to a final volume of 4.5 mL. Inject into preparative HPLC        to collect product peak.    -   5. Freeze dry the collected fraction overnight (at least 15        hour).    -   6. Weigh the product and transfer to a brown bottle. Store in a        desiccated bag at −20° C.        Typical yield=12 mg        Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-SATA-Alc: elution time = 27.6 min m⁺/e = 559.1 and 838.5Preparation of Os-SMCC-Alc Conjugate

-   -   1. Weigh 39.0 mg Os-SMCC (0.0452 mmol) and transfer to a 5 mL        Reacti-Vial with stir bar. Add 1.0 mL PBS (pH 6.0) to dissolve.    -   2. Add 30.0 mg Hblc-Cys peptide (0.0450 mmol). Let reaction        proceed at RT for 2 hours. Inject a sample into analytical HPLC        to monitor reaction progress.    -   3. If reaction is complete, dilute the mixture with 0.1% TFA        buffer to a final volume of 4.5 mL. Inject into preparative HPLC        to collect product peak.    -   4. Freeze dry the collected fraction overnight (at least 15        hour).    -   5. Weigh the product and transfer to a brown bottle. Store in a        desiccated bag at −20° C.        Typical yield=12 mg        Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-SMCC-Alc: elution time = 27.6 min m⁺/e = 480.5 and 534.4Preparation of Os-SMCC-Ao Conjugate

-   -   1. Weigh 37.0 mg Os-SMCC (0.0426 mmol) and transfer to a 5 mL        Reacti-Vial with stir bar. Add 1.0 mL PBS (pH=6.0) to dissolve.    -   2. Add 24.3 mg HbAo-Cys peptide (0.0425 mmol). Let reaction        proceed at RT for 2 hours. Inject a sample into analytical HPLC        to monitor reaction progress.    -   3. If reaction is complete, dilute the mixture with 0.1% TFA        buffer to a final volume of 4.5 mL. Inject into preparative HPLC        and collect the product peak.    -   4. Freeze dry the collected fraction overnight (at least 15        hour).    -   5. Weigh the product and transfer to a brown bottle. Store in a        desiccated bag at −20° C.        Typical yield=15 mg        Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-SMCC-A_(o): elution time = 27.9 min m⁺/e = 360.7 and 720.5Preparation of Os-DSG-Alc Conjugate

-   -   1. Weigh 32.0 mg Os-DSG (0.037 mmol) and transfer to a 5 mL        Reacti-Vial with stir bar. Add 0.75 mL DMF to dissolve. Add 25        μL triethylamine.    -   2. Add 26.5 mg HbAlc-Lys peptide (0.0349 mmol). Let reaction        proceed at RT for 2 hours. Inject a sample into analytical BPLC        to monitor reaction progress.    -   3. If reaction is complete, dilute the mixture with 0.1% TFA        buffer to a final volume of 4.5 mL. Inject into preparative HPLC        and collect the product peak.    -   4. Freeze dry the collected fraction overnight (at least 15        hour).    -   5. Weigh the product and transfer to a brown bottle. Store in a        desiccated bag at −20° C.        Typical yield=16 mg        Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-DSG-Alc: elution time = 23.5 min m⁺/e = 501.8 and 752.8Preparation of Os-DSG-Ao Conjugate

-   -   1. Weigh 52.0 mg Os-DSG (0.0605 mmol) and transfer to a 5 mL        Reacti-Vial with stir bar. Add 1.0 mL DMF to dissolve. Add 25 μL        triethylamine.    -   2. Add 49.1 mg Fmoc-HbA₀ peptide (0.0606 mmol). Let reaction        proceed at RT for 2 hours. Inject a sample into analytical HPLC        to monitor reaction progress by the appearance of peak at 40.3        min for Os-DSG-A₀(Fmoc).    -   3. If reaction is complete, inject additional 100 μL        triethylamine. After I hour, inject sample into analytical HPLC        to determine whether all Fmoc protection group is removed by        disappearance of the peak at 40.3 min.    -   4. If removal of Fmoc is complete, dilute the mixture with 0.1%        TFA buffer to a final volume of 4.5 mL. Inject into preparative        HPLC to collect product peak.    -   5. Freeze dry the collected fraction overnight (at least 15        hour).    -   6. Weigh the product and transfer to a brown bottle. Store in a        desiccated bag at −20° C.        Typical yield=16 mg        Product is analyzed by HPLC and ES/MS.

HPLC ES/MS Os-DSG-A_(o): elution time = 23.2 min m⁺/e = 447.4 and 670.3

Synthesis of bis(4,4′-dimethyl-2,2′-bipyridyl)4-methyl-4′-carboxylpropyl-2,2′-bipyridyl osmium[Os(dm-bpy)2(mcp-bpy)]Cl2

Potassium hexachloroosmium was reacted with 4,4′-dimethyl-2,2′-dipyridylat 1:2 molar ratio by refluxing in DMF. The potassium chlorideprecipitate was filtered and the dimethyl-bipyridyl dichloroosmiumcomplex was reduced from +3 oxidation state to +2 oxidation state usingexcess sodium dithionite. The product was recrystallized in DMF/watermixture at 0° and recovered by filtration.

4,4′-Dimethyl-2,2′-bipyridyl dichloroosmium was reacted with4-methyl-4′-carboxylpropyl-2,2′-dipyridyl by refluxing in ethyleneglycol. The solvent was removed by rotary evaporation. The product wasdissolved in DMF and precipitate in ethyl ether. The product was driedin a vacuum oven overnight.

Analyticals: Product and intermediate product were analyzed by HPLC andmass spectroscopy for purity and identity of the compound.

Os(dm-bpy)2Cl2: Theoretical MW=629.6, MS showed 8 isotope peaks withmost abundant peak at 630. HLPC elution time at 29.94 min with a purityof 90%+

Os(dm-bpy)2(mcp-bpy): MS confirmed the MW at 814.5 and HPLC showed apurity greater than 85%.

Synthesis of biotin-Os Complex Conjugate Biotin-Os(dm-bpy)2(mcp-bpy)Cl2

The carboxyl group was activated by reacting the above Os complex withdicyclohexylcarbodiimide in the presence of N-hydroxysuccinimide. Theactive ester Os complex was isolated using preparative HPLC method andthen reacted with amine-containing biotin to form the final conjugate.

Experiment to Independently Measure the Concentration of TwoElectroactive Conjugate Species

Os(bipy)HisCl-DSG-HbAlc was prepared as described above.

Ferrocene-AMCHA-DADOO-biotin was prepared from ferrocene monocarboxylicacid, the crosslinker aminomethylcyclohexylic acid, the chain extender1,8-diamino-3,6-DiOxoOctane and biotin as described elsewhere.

Mixtures of the two conjugates were prepared to evaluate the ability ofthe method of the invention to independently measure the concentrationof the conjugates, and make corrections for variations in reagentamounts, electrode response, and environmental conditions.

Part 1: Simple Mixed Conjugate Response

The following matrix of solutions was prepared in 10 mM phosphate bufferwith 150 mM NaCl and 0.5% Brij-35, a non-ionic surfactant.

Os-DSG- Ferrocene- Os-DSG- Ferrocene HbAlc Biotin HbAlc Biotin uM/l uM/luM/l uM/l 0 0 0 12.5 6.25 0 6.25 12.5 12.5 0 12.5 12.5 25 0 25 12.5Os-DSG- Ferrocene- Os-DSG- Ferrocene HbAlc Biotin HbAlc Biotin uM/l uM/luM/l uM/l 0 25 0 50 6.25 25 6.25 50 12.5 25 12.5 50 25 25 25 50

An Interdigitated array (IDA) microelectrode was fabricated according tothe procedures described. In addition to the IDA, the chip had asilver/silver chloride electrode on the surface to function as thereference electrode and counter electrode. This electrode was producedwith the same lithographic process, and then electroplated with silverand silver chloride according to standard techniques. The IDA wasconnected to a bipotentiostat capable of controlling the potentialrelative to the reference and measuring the current at each of theelectrodes of the IDA. Aliquots of the solutions were placed onto thesurface of the chip, such that the IDA and the reference electrodes werecovered.

Measurements were made by first applying −100 mV (vs. Ag/AgCl) to oneelectrode of the IDA, and 200 mV to the other electrode for a period of30 seconds. At this time, current was measured at each electrode. Then200 mV was applied to one electrode, and 550 mV to the other. After 30seconds, current was again measured. See FIG. 9 for a summary of theresults, which clearly demonstrate that the concentrations of the twomediators can be independently measured with this method.

Part 2. Concentration Co-variance of Dual Mediators on IDA Electrodes

In this experiment, it was demonstrated that by making a mixture ofknown concentrations of two different mediators, and measuring differentdilutions of that mixture by the method of the invention, the ratio ofthe concentrations of the mediators remains constant. (Internal standardapplication).

The same two mediator conjugates were used as in Part 1. (Os-DSG-Alc andFc-Bi)

From a solution containing 40 uM of each conjugate, solutions containing27 uM, 32 uM, and 36 uM of each conjugate were prepared in the samebuffer (PBS/Brij).

The solutions were measured as in the previous example. Each solutionwas measured on 5 different IDA electrodes. The results are presented asthe means, standard deviations, and coefficient of variation for eachsolution separately, and for all solutions pooled over all electrodes.

Individual Os-DSG- concentrations Alc Fc-Bi Os/FC 28 uM Mean 156 85 1.84S.D. 17.5 13.2 0.08 % C.V. 11.2 15.5 4.4 32 uM Mean 165 88 1.88 S.D. 117.2 0.04 % C.V. 6.7 8.2 2.0 36 uM Mean 189 102 1.85 S.D. 12.5 6.9 0.04 %C.V. 6.6 6.7 2.3 40 uM Mean 208 110 1.90 S.D. 9.5 5.5 0.06 % C.V. 4.65.0 3.4 Pooled Concentrations Mean 182 97 1.87 S.D. 24.4 13.2 0.06 %C.V. 13.4 13.5 3.4

This example clearly demonstrates that the internal standard effect ofmeasuring two conjugates or mediators and calculating the ratio givessignificantly improved precision of measurement, not only within eachsolution (compensation for variation between electrodes) but over allsolutions (compensation for variation in sample dilution or amount).

Part 3. Temperature Compensation

It was desired to show the effectiveness of the method in compensatingfor environmental influences such as Temperature variation on theaccuracy or the measurement.

The same two conjugates were prepared in solution at 40 uM as before.They were measured as before on IDA electrodes, either at roomtemperature or warmed to 35–40 C. on a heated metal plate prior to themeasurement. The solutions were also warmed to 37 C. prior toapplication to the electrodes.

Room Temperature Warmed Ratio of response (23 C.) (35–40) C. Warm/RTOs-DSG-Alc 261 387 1.45 Fc-Bi 179 268 1.5 Ratio Os/Fc 1.46 1.44 0.99

As demonstrated by the results, the measured values increase by almost50% in the case of the warmed samples, which would lead to a largemeasurement error. However the use of the internal standard and ratiocalculation effectively eliminates the temperature dependence of theresult.

Immunoassay Detection of HbAlc with Osmium Mediator Conjugates

The goal of all diabetic therapy is to maintain a near normal level ofglucose in the blood. Home blood glucose kits are available to monitorthe current glucose values and are valuable for diabetics to adjust dayto day insulin doses and eating habits. Unfortunately, since the testsonly measures a point in time result, it does not tell them the overalleffectiveness of their actions in maintaining glycemic control.Measurement of glycosylated hemoglobin is now becoming widely acceptedas an index of mean blood glucose concentrations over the preceding 6–8weeks and thus provides a measure of the effectiveness of a diabetic'stotal therapy during periods of stable control. Since monitoring adiabetic's glycated hemoglobin can lead to improved glycemic control,the ADA recommends routine measurements of four times a year up to oncea month for less stable type I diabetics.

Several technologies are available for the measurement of glycatedhemoglobin. They include immunoassays for HbAlc (TinaQuant, BMC;DCA2000, Ames; and Unimate, Roche), ion exchange (Variant, BioRad; EagleDiagnostics), and affinity chromatography (ColumnMate, Helena; GlyHb,Pierce).

One objective of this project is to develop a simple to use disposablestrip for electrochemical detection of HbAlc for use in both physicianoffices and the home.

The most significant parameter for assessing patient condition is ratioof HbAlc to HbA₀, and thus the measurement of both glycated (HbAlc) andnonglycated (HbA0) values is required to calculate the ratio. Thisrequires two separate measurements. It is preferable to use the sametechnology to measure both the glycated and nonglycated fractions, thusremoving some sample and environmental interferences. Measurement ofHbAlc via electrochemical immunoassay is described below.Electrochemical HbA0 immunoassay measurements are carried out using thesame methods as that for HbAlc. The concentrations of HbA₀ aresignificantly higher. One alternative to A₀ measurements usingimmunoaffinity would be to measure total hemoglobin directly usingbiamperometry or differential pulse voltammetry. This can be easilyaccomplished since hemoglobin is readily oxidized by[0s(bpy)₂(im)Cl]²⁺Cl₂.

The N-terminal valine of the β-chain of hemoglobin A is the site ofglycosylation in HbAlc, and serves as a recognition site for theantibody. In whole blood the N-terminal valine is not accessible for theantibody to bind. Access is gained by lysing the red cells to releasethe hemoglobin followed by a conformation change (denaturing orunraveling) to adequately expose the HbAlc epitope. Dilution of thesample may occur as part of the lysing/denaturing process or may berequired post denaturing to prepare the sample for the antibody (adjustpH, other) or bring the sample into a range suitable for electrochemicalimmunoassay. In one embodiment, a fixed amount of antibody is incubatedwith the prepared sample and it binds to the HbAlc epitopes of thesample. The free antibody and the antibody bound sample is then combinedwith the osmium peptide conjugate (Alc or A₀) to allow the remainingunbound antibody to bind to the mediator label. When the mediator isbound to the antibody (a macromolecule), it can not freely diffuse tointeract with the electrode and thus currents generated aresignificantly reduced. The remaining unbound mediator label is thereforeproportional to the concentration of HbAlc in the sample. The unboundmediator can be measured electrochemically either through an enzymeamplification method or directly using an interdigitated array electrodewith bipotentiostatic control.

An electrochemical HbAlc immunoassay response was demonstrated on goldelectrodes using enzyme amplification in a biamperometric mode. Theexperimental conditions were not thoroughly optimized and the assaycomponents were premixed in microcentrifuge tubes and then pipette ontothe gold 4.95 mm² E-cells. The experimental conditions for this assayare shown in Table 1. Table 2 shows the dose response data for the low,medium, and high HbAlc samples. FIG. 4 shows the dose response with twoadditional points LL and HH which represent a diluted low and aconcentrated high sample. The higher currents for LL may possibly beexplained by a reduction in blood proteins (due to dilution) leading toa reduction in electrode fouling.

TABLE 1 Dose Response Conditions Electrochemical Sample Preparation(denaturing) Reagents Measurement 1. 20 μL Lysed blood for L, M, H 1. 50μL 12.5 μM PAB 1. VXI Waveform samples (previously frozen wholeIS<Alc>in PBST E-450 mV for 60s blood 2. 50 μL denatured Blood 2. InsertGold Electrodes 2. 18 μL of L (LL) and 22 μL of H for 3. 20 μL 40 μMOs-DSG- WE = 1.5 × 3.3 mm (HH) Alc in DI H₂O 3. Apply 20 μL Reagent 3.480 μL 1.5 M LiSCN with 0.1% 4. 20 μL 1M Glucose in 4. Startbiamperometric Tween DI H₂O measurement mode 4. Mix and allow todenature for 10 5. 20 μL Glucdor/PQQ 5. Extract 60 sec data minutes(Vortex) (1.7 mg/ml/0.17 mg/ml)

TABLE 2 Blood Dose Response Data Separate denaturant step All Data g/L#1 #2 #3 #4 Mean SD CV Mean SD CV Current @ 60 seconds (nA) 6.49 76.2878.34 84.79 78.47 79.47 3.69 4.64 81.54 4.67 5.73 6.49 80.30 82.97 86.8283.48 83.48 2.69 3.22 6.49 83.25 85.49 90.05 86.67 86.67 2.94 3.40 6.4976.66 71.72 78.24 76.56 76.55 1.44 1.88 12.34 95.61 95.05 93.06 94.0294.44 1.13 1.20 98.31 4.19 4.26 12.34 98.59 106.29 103.81 102.08 102.083.60 3.53 12.34 95.80 103.14 99.90 99.32 99.32 3.06 3.08 12.34 93.1696.2 103.10 97.41 97.41 4.16 4.27 18.20 100.06 110.83 119.08 112.52112.52 9.29 8.26 113.48 5.08 4.48 18.20 115.43 116.93 116.77 116.26116.26 0.71 0.61 18.20 116.12 109.72 112.07 112.21 112.21 2.78 2.4818.20 118.13 112.16 109.8 112.93 112.93 3.61 3.20 Calculated HbAlc(g/dL) 6.49 4.46 5.22 7.58 5.26 5.63 1.35 24.00 6.39 1.71 26.80 6.495.94 6.91 8.33 7.23 7.10 0.99 13.87 6.49 7.02 7.84 9.51 8.72 8.27 1.0813.05 6.49 4.60 3.89 5.18 4.56 4.56 0.53 11.58 12.34 11.55 11.34 10.6110.97 11.12 0.41 .3.72 12.54 1.54 12.25 12.34 12.64 15.46 14.56 13.0313.92 1.32 9.48 12.34 11.62 14.31 13.12 12.58 12.91 1.12 8.70 12.3410.65 11.77 14.29 12.12 12.21 1.53 12.50 18.20 13.18 17.13 20.15 20.5417.75 3.41 19.19 18.10 1.86 10.29 18.20 18.82 19.37 19.31 19.00 19.120.26 1.36 18.20 19.07 16.72 17.58 17.16 17.63 1.02 5.78 18.20 19.8117.62 16.75 17.42 147.90 1.32 7.40 n = 16, 4 denaturation for each HbAlcsample × 4 replicates each, Y = 2.82 × + 62.54, See fie 4435051 A.xls

Blood lysis is necessary to release the hemoglobin followed bydenaturing to expose the HbAlc epitope. Lysis can easily be accomplishedvia surfactants, osmotic effects of dilution with water, and directly bymany denaturants. Blood lysed through a freeze/thaw cycle was shown notto significantly interfere with the biamperometric measurement (“openrate” with and without lysed blood was almost identical). Conversely,denaturing the lysed blood with a variety of known denaturants to exposethe HbAlc epitope has shown significant suppression of theelectrochemical response, inhibiting measurement of an HbAlc doseresponse. Only LiSCN and citric acid from the list of evaluateddenaturants shown in Table 3 was able to expose the Alc epitope andminimize protein fouling enough to measure an HbAlc dose response.

Denaturing the sample for antibody recognition without severely foulingthe electrode surface is important for successful development of anHbAlc immunoassay. Although LiSCN has been used almost exclusively toshow feasibility, it has many limitations that would hinder its use inthe disposable. Citric acid, a solid at RT may offer benefits as adenaturant if it could be dried onto a strip followed by a diluent toadjust the pH to neutral. Acid or base blood denaturing followed by afinal pH adjustment with a buffered diluent is an area worth furtherevaluation. One problem that was initially encountered was precipitationin adjusting the pH back to neutral, which can be overcome by using adifferent buffer or with the addition of surfactants.

TABLE 3 Blood Denaturants Method Comments KSCN Initial work did not showa dose response with KSCN denatured blood. KSCN has a larger negativeeffect on the electrochemical response than LiSCN. Literature shows thatLiSCN is more effective than KSCN (concentrated efforts on LiSCN).DCA2000 Denaturing of blood not evident with the higher blood Bufferconcentrations required for this assay. Higher concentrations of LiSCNare shown below. LiSCN Method used by Ames DCA2000 HbAlc immunoassay.Citric, Sulfuric, Blood HbAlc dose response (high/low) was seen withHydrochloric, citric acid and was comparable to the response with &Perchloric LiSCN Acid Evidence of blood denaturing was seen by all:“solution turned brown.” Citric acid is preferred. Adjustment of pH toneutral after denaturing also saw problems of precipitation. Enzymemediated responses with Gluc-Dor at pH 5.7 reduces response 50% comparedto pH 7–8. Citric acid blood denaturing method is shown in FIG. 5.Pepsin/Citric Roche HbAlc immunoassay uses pepsin/citric acid to Acidhemolyze and proteolytically degrade hemoglobin to glycoproteinsaccessible by the antibody. Denaturation was apparent by the colorchange to a brownish red solution. Hemoglobin Alc dose response(high/low) was obtained comparable to LiSCN and citric acid denaturants.The procedure was identical to that of citric acid used above with theexception of pepsin added to the acid. Results were identical to that ofcitric acid. TTAB (Tetra Method of denaturing used in the TinaQuantHbAlc decyltrimethyl turbidimetric immunoassay. ammonium Evidence ofdenaturing: “solution turned green” bromide) TTAB concentrations0.0125–0.2% severely suppressed enzyme mediated (Glucdor/PQQ/Glucose)biamperometric measurements. Open rates were 16–50 nA compared to 140 nAwithout TTAB. NaOH Evidence of denaturing: “solution turned brown.” NaOHdoes not adversely effect the enzyme mediated electrochemistry. Even athigh pH the open rates do not change, although pH adjustment willprobably be required to bring it within an optimal range for theantibody. NaOH denatured blood suppresses the open rates probably due toprotein fouling. Lowering the pH to neutral tends to cause someprecipitation.

TABLE 4 Effective Blood Denaturing Procedure for 2% Blood LiSCN (OneStep) LiSCN (Two Step) Citric Acid (2 Step) 40 μL 6M LiSCN 960 μL 1.5MLiSCN 200 μL 0.2M Citric Acid 20 μL 5% Tween in PBS 20 μL 5% Tween inPBS 20 μL 5% Tween in PBS 20 μL Blood 20 μL Blood 20 μL Blood Mix(vortex) and allow to denature Mix (vortex) and allow to denature Mix(vortex) and allow to for 10 minutes. Dilute with 920 μL for 10 minutes.denature for 10 minutes. Dilute DI H₂O. with 760 μL 8X PBS (0.1% Tween)Denaturing time was not optimized. Denaturing time was not optimized. Nooptimization studies were Limited data supports longer times Dataindicates shorter times may be performed. for better precision usingthis adequate. method. PBS = 10 mM Phosphate Buffer, 2.7 mM KCl, 137 mMNaCl pH = 7.4 Increased level of surfactant (5% Tween) reduces oreliminates precipitate.

Electrode fouling caused by denatured blood proteins adsorbing to theelectrode surface can impede electron transfer and thus decreaseelectrode sensitivity. Electrode fouling or passivation occurs more orless immediately following sample contact with the surface thusminimizing the severity of denaturing in the sample should be the firstapproach. Surface conditions that are hydrophobic will favor adhesion ofthe proteins and thus fouling may be minimized with electrode surfacesof higher surface energies. This explains why gold electrodes shows lessfouling with denatured blood than palladium. Reduction of proteinfouling may be achieved by changing or protecting the electrode surface.Modifications that make the surface more hydrophilic should reduce theamount of fouling and can be accomplished by argon or oxygen plasmatreatment or corona treatment. Selective coatings that could block theproteins from reaching the electrode surface can usually partiallycircumvent the problem have been used in the field to reduce fouling.Unfortunately, dramatic decreases in responses greater than seen withthe denatured blood proteins are normally noted with their use.Hydrophilic coatings such as PEO were also evaluated and showed someimprovement, but have similar problems of decreased magnitude andprecision caused by forcing reagents to diffuse through the polymers.Reagents dispensed and dried over the electrodes may help reduce themagnitude of protein fouling with less negative effects.

Mediator concentration dose response, inhibition with antibody andreversal with a BSA-Alc polyhaptan were evaluated and summarized inTable 5. The Os-DSG-Alc is stable in a lyophilized form and when frozenin solution at −20° C. (40 and 80 μM).

TABLE 5 Osmium Mediator Labels Mediator Concentration Inhibition withLabel Response Antibody Reversal Comments Os-SMCC- Linear PAB IS (≦92%)Yes with BSA- % = Inhibition values ranged Alc PAB DE (≦97%) Alcpolyhaptan from 16% to 97% depending MAB (≦50%) on age of Os-SMCC stocksolution. Degrades in solution. Os-SATA- Linear PAB IS (≦44%) Yes withBSA- Stability similar to SMCC. Alc Alc polyhaptan Os-DSG-Alc Linear PABIS (≦91%) Yes with BSA- More stable than conjugate PAB DE (≦87%) Alcpolyhaptan made with SATA and SMCC MAB (≦78%) crosslinker but stilldegrades in solution. Os-SATA-A₀ Linear Yes with Sheep No with A₀HB- A₀conjugate was found to be B<HbA₀> (≦84%). peptide#1 unstable insolution. No with Zymed Conjugate was not rabbit antibodies lyophilized.

Polyclonal DE (ion-exchange) purified sheep antibody is used in theTinaQuant HbAlc assay. IS (immunosorbent) antibody is prepared usingstandard IS purification methodology. Samples of a monoclonal antibodywere also obtained for evaluation. Inhibition curves were performed insolution with all mixing occurring in microcentrifuge tubes. Assays weremeasured by applying 20 μL onto 6 mm² palladium electrodes with theconditions shown in Table 5. Inhibition curves with the three hemoglobinAlc antibodies (PAB IS, PAB DE, and MAB) were generated by fixingOS-DSG-Alc at 5 μM and varying the antibody concentration. Both PAB ISand MAB showed the expected stoichiometric relationship for inhibitionwith the osmium peptide conjugate indicating efficient and fast bindingof the antibody to the Alc peptide. The polyclonal IS and monoclonalboth showed steep inhibition curves with maximum change being reachedclose to 5 μM. Additional antibody above 5 μM showed little effect onincreasing the inhibition. The less purified PAB DE antibody had a muchsmaller slope and as expected required more than 3 times the amount toget close to maximum inhibition. FIG. 6 shows the inhibition curves foreach of the HbAlc antibodies tested. From the inhibition curves we wereable to select reasonable concentrations of antibody for maximumreversal with Alc samples.

Inhibition curves were also performed for the Os-SMCC-Alc (Max=97%) andOS-SATA-Alc (Max=44%) mediator labels. Stability of the mediator labelswere also evaluated by monitoring % inhibition values over time. All ofthe mediator labels showed some degradation when stored in dilutesolutions (40 μM) at RT. Samples frozen at −20° C. appear to be stable.

For demonstrating inhibition reversal, antibody concentrations of 4 μMfor both PAB IS and MAB and 15 μM for PAB DE were chosen from theinhibition curves shown above. Reversal curves were then generated usinga series of dilutions of BAS-Alc polyhaptan with a ˜1:1 Alc:BSA. TheBSA-Alc acts as our sample and binds to the antibody. FIG. 10 shows thereversal curves for the three antibodies.

While these feasibility studies for a HbAlc immunoassay used an enzymemediated amplification method. (Glucdor/PQQ/glucose was used toregenerate reduced mediator after oxidation at the electrode surfaceproviding a higher diffusion controlled current is given by the cottrellequation), they are considered to be indicative of results attainablewith the use of IDA electrodes with bipotentiostatic control inaccordance with this invention.

1. A method for measuring the concentration of an analyte in a sample ofa liquid, said method comprising contacting said sample withpredetermined amounts of (1) a specific binding partner for saidanalyte, and (2) a detectable compound of the formula:

wherein R and R₁ are the same or different and are 2,2′-bipyridyl,4,4′-disubstituted-2,2′-bipyridyl, 5-5′-disubstituted,-2,2′-bipyridyl,1,10-phenanthrolinyl, 4,7-disubstituted-1,10-phenanthrolinyl, or5,6-disubstituted-1,10-phenanthrolinyl, wherein each substituent is amethyl, ethyl, or phenyl group, R₅ is 4-substituted-2,2′-bipyridyl or4,4′-disubstituted-2,2′-bipyridyl wherein the 4-substituent is the groupB-(L)_(k)-Q(CH₂)_(i)- and the 4′-substituent is a methyl, ethyl orphenyl group; R, R₁ and R₅ are coordinated to Os through their nitrogenatoms; Q is O, S, or NR₄ wherein R₄ is hydrogen, methyl or ethyl; —L— isa divalent linker; k is 1 or 0; i is 1, 2, 3, 4, 5 or 6; B comprises aligand that binds to the specific binding partner and is selected sothat the detectable compound binds competitively with the analyte tosaid binding partner; d is +2 or +3; X and Y are anions selected frommonovalent anions chloride, bromide, iodide, fluoride,tetrafluoroborate, perchlorate, and nitrate and divalent anions sulfate,carbonate or sulfite wherein x and y are independently 0, 1, 2, or 3 sothat the net charge of X_(x)Y_(y) is −2 or −3; and determiningelectrochemically the concentration of the detectable compound not boundto the specific binding partner, wherein the concentration of thedetectable compound not bound to the specific binding partner isproportional to the concentration of analyte present in the sample offluid.
 2. The method of claim 1 wherein the liquid sample is dilutedblood, the analyte is glycosylated hemoglobin and the group B in thedetectable compound comprises a glycosylated peptide of the formulaGluc-Val-His-Leu-Thr (SEQ. ID NO. 1).
 3. The method of claim 2 furthercomprising the step of determining the concentration of total hemoglobinor unglycosylated hemoglobin in the liquid sample, and calculating theratio of the concentration of glycosylated hemoglobin to unglycosylatedhemoglobin.
 4. The method of claim 1 wherein the concentration of theunbound detectable compound is determined by dc voltammetry using aninterdigitated microarray electrode.